harvesting RNA from silique tissue

Tamara Western western at mail.botany.ubc.ca
Mon Dec 16 15:39:37 EST 2002


<x-flowed>Hi all,

     I have already emailed Julia personally, but I guess I'll wade into
the public forum as well. I have spent some time looking for and testing
protocols to isolate RNA from siliques at various developmental stages
to use for microarray analysis. Here is what I learned:  First, as a
couple of people have stated, Trizol does not work for siliques. In
fact, guanidium-based preps seem not to work well for siliques, except
when they are very young. (This was noted by a couple of labs in a
previous newsgroup discussion on silique preps & I've seen it fail in
several hands, including my own). Second, though the Qiagen RNeasy kit
works fine for various plant tissues, we have found that it does not
work for siliques except when they are very young (it is also
guanidium-based).  I have, however, found RNeasy extremely useful for
doing a secondary purification to get very clean RNA after I have
isolated it from siliques.
     The prep that I ended up with is a variation on the old-fashioned
acid phenol-LiCl prep. The original version of the prep I have came from
Jerome Giraudat's lab where they used it very successfully to do
Northerns on all stages of seed development from fertilization to dry
seeds. By comparison to another acid phenol-LiCl prep that a colleague
of mine got elsewhere, it seems to be pretty standard, with the
exception of an emphasis on the slow addition of the LiCl with gentle
vortexing. To this, I have appended a wash of the pellet with sodium
acetate to get rid of excess polysaccharides (recommended by another
colleague who used such a wash to remove polysaccharides from RNA from
cultured cells). And, finally, for the microarray RNA, I did secondary
purification by running the RNA through Qiagen RNeasy columns. My yield
is about 0.5 ug/mg silique tissue (after the columns), with A260/280 of
1.96-2.00, the RNA looks great on a gel & works for 1-step & 2-step RT-PCR.

cheers, Tamara

PS I'd be happy to send my protocol to anyone who wants it, just drop me
an email.

PPS   There is also a protocol from the Ohlrogge lab for isolation of
RNA from developing seeds for microarrays that was published in
Biotechniques (Ruuska & Ohlrogge. 2001. BioTechniques 31:752-758). It is
extremely long and complicated and I tried it, but I ended up with
mostly degraded RNA. Since I had another prep that worked and this one
was a lot of effort, I never bothered to trouble-shoot it. It is also
worth considering since it works well for them.

-- 
Dr. Tamara L. Western
Research Associate
Department of Botany and Biotechnology Laboratory
University of British Columbia
6270 University Blvd., Vancouver BC  V6T 1Z4
Phone: 604-822-2437; Fax: 604-822-6089
Email: western at mail.botany.ubc.ca



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